Jpn. J. Human Genet. 36, 195-199, 1991
DNA ANALYSIS DIFFERENT
OF A PATIENT
MARKER
WITH
CHROMOSOMES
TWO USING
Y-SPECIFIC DNA PROBES
Takashi TAMURA,1,2 Yoshikazu KUROKI,8 Shigeo NAGAFUCHI,1 Seizo SUWA,4 Yutaka NAKAHORI,1 K a z u m i t s u TERASttlMA,5 Toshiyuki FuRusuofl and Yasuo NAKAGOME1 1Department of Congenital Abnormalities Research, National Children's Medical Research Center, Setagaya-ku, Tokyo 154, Japan 2Department of Clinical Genetics, Kyorin University School of Heahh Sciences, Mitaka, Tokyo 192, Japan 3Division of Medical Genetics, 4Department of Endocrinology and ~Department of Urology, Kanagawa Children's Medical Center, Minami-ku, Yokohama 232, Japan
Summary A female patient with unilateral gonadal dysgenesis was a mosaic for three cell lines, 45,X/46,X,+marI/46,X,+marII, including two different marker chromosomes. DNA analysis using 17 Y-specific DNA probes revealed that each marker consists of different segments of the Y chromosome. Key Words Y chromosome, marker chromosome INTRODUCTION
It is difficult to identify the origin of small marker chromosomes using cytological techniques as many marker chromosomes fail to show characteristic banding patterns. In the present report, we used 17 Y-specific DNA probes to analyze DNA from a female patient with clitoral enlargement who had two different marker chromosomes. Each of the markers consists of a part of the Y chromosome. CASE REPORT
A newborn girl was investigated, because of clitoral hypertrophy and bilateral inguinal hernias. The presence of vagina and uterus was established by the urethrography. Clitoroplasty and surgery to correct the bilateral inguinal hernias were Received March 2, 1991; revised version received May 1, 1991; Accepted May 7, 1991. 195
196
T. TAMURA et aL
performed. The right hernial content was proven to be testis and was excised. A uterus and a possible streak gonad were found in the left inguinal hernia and a small part of the latter was removed. At eight months of age, no development delay was apparent. Clinical features were normal except for those noted above. Laboratory analyses revealed: blood testosterone 0.9 ng/ml; elevated LH (282 ng/ml) and F S H (655 ng/ml), normal values being 0-1, 4-63 and 0-150 respectively; and normal adrenal function. CYTOGENETIC STUDIES A 4 5 , X / 4 6 , X , + m a r I / 4 6 , X , + m a r I l karyotype was observed in cells cultured from peripheral blood leukocytes (Fig. 1). Out of 31 cells karyotyped, five were 45,X, 23 had mar I and three had mar II. Both markers I and II were Q-negative. The former was a small ring chromosome (Fig. la). The latter was a G-sized chromosome. In some of mitotic spreads marker II appeared to be dicentric (Fig. lb). Additional material for a C-band study was not available. DNA STUDIES A total of 17 Y-specific D N A probes were used. Ten of them: 87-4(AMGL); 87-6(DYS135); 87-7(DYSI30); 87-17(DYS131); 87-19(DYS132); 87-24(DYS133); 87-26(DYS139); 87-27(DXYS73Y); 87-28(DYS140), and 87-31(DYS134), were cloned by Nakahori et al. (1991). The pHY10 probe detects the repetitive D N A family DYZ1 in the Q-positive region (interval 7) of the Y chromosome (Nakahori
Fig. 1. Two marker chromosomes in the patient. Top: Partial karyotype of a cell with marker I. From left; G stained chromosome pairs, numbers 21 and 22, an X chromosome and marker I. At the far right, the Q-stained marker is shown. Bottom: As above except that marker II instead of marker I is shown. Jpn. J. Human Genet.
TWO DIFFERENT MARKERS
50f2
197
pHY10
Fig. 2. Examples of Southern analysis. For each probe, both control male DNA and that of the patient are shown. In the 50f2 blot (left), arrow heads indicate loci A to E (from the top). Loci A, B and D showed decreased but distirlct signals ( + § in Fig. 3). The band corresponding to locus C of S0f2 barely distinguishable. The band corresponding to 50f2 locus E is not detectable under our standard hybridization conditions and was observed only after prolonged exposure (not shown). The patient is pHY10 negative (right). et al., 1986). The remaining six probes were gifts from Dr. J. Weissenbach (probes 47z, 52d, 50f2, 12f3, and 49f; Vergnaud et al., 1986) and a gift from Dr. D. C. Page (probe pDP1007(ZFY); Page et al., 1987). Probe 50f2 detects five different loci (50f2A to 50f2E) on the Y chromosome (see Fig. 2). Probe 52d detects three different loci: 52dA ( Y q l l ) ; 52dB (Yp) and 52dC (Yp). Probe 87-17 (DYS131) detects two different sites DYS131A (Yq) and DYS131B (Yp). The 17Y-specific D N A probes had already been mapped on the Y chromosome and were found to detect a total of 23 loci (Nakahori et aL, 1991). D N A was isolated from peripheral leukocytes by the standard technique (Maniatis et al., 1982). Probes pDP1007, 47z, 52d, 87-4, 87-7, 87-19, 87-26, and Vol. 36, No. 2, 1991
198
T. T A M U R A et al.
87-27 were used to probe Southern blots of TaqI digested patient DNA. The pHY10 probe was used in combination with StuI (Nakahori et aL, 1989). The remaining probes were used with EcoRI. Differences were observed between the patient DNA patterns of probes from separate regions of the Y chromosome. Firstly, the Y-specific repeated sequence DYZl identified by pHY10 was absent in the patient (Fig. 2). Second, the intensities of the signals produced, when the other probes were hybridized to the patient DNA, varied according to the location of those probes on the Y chromosome. Medium signal intensities were observed for probes between pDP1007 in interval 1 and 87-19 in interval 5-6. Low signal intensities were observed for probes between 87-6 and 49f in interval 5-6. The results of the DNA analysis are summarized in Fig. 3.
Probes/ loci pDP1007 47z 87-27 52dB 52dC 87-17B 50f2A,B
-IF "H-H44-H-IF -H-
50f2D
-IF
87-7 87-4 12f3 87-28 87-26 87-24 87-31 87-19 87-6 52dA 87-17A 50f2E 50f2C 49f pHYlO
+t-H4-I-i+ 44-H-H"H-t+ --t+ + +
Fig. 3.
1!
patient
Pseudoautosoma
1~ 2
i
"--] ,3~
n Sex9 . ~ IIFIK OQ
pDPlOO7
87-27(DXYS73Y) ;S~C~
5807;217B(DYS 131'
i~il " - ] 4 A J [--50,2D' ~ 1 I 1 8 7 - 7 (DYS130) ~ _ _ 1 4 B A--12f3 L_ 87_4 (AMGL) f~.:iiiil.---~ J ~87-28(DYS140) [~iiii] 1 5 , 87-26(DYS 139) [~iiit/I ~:
DNA ANALYSIS DIFFERENT
OF A PATIENT
MARKER
WITH
CHROMOSOMES
TWO USING
Y-SPECIFIC DNA PROBES
Takashi TAMURA,1,2 Yoshikazu KUROKI,8 Shigeo NAGAFUCHI,1 Seizo SUWA,4 Yutaka NAKAHORI,1 K a z u m i t s u TERASttlMA,5 Toshiyuki FuRusuofl and Yasuo NAKAGOME1 1Department of Congenital Abnormalities Research, National Children's Medical Research Center, Setagaya-ku, Tokyo 154, Japan 2Department of Clinical Genetics, Kyorin University School of Heahh Sciences, Mitaka, Tokyo 192, Japan 3Division of Medical Genetics, 4Department of Endocrinology and ~Department of Urology, Kanagawa Children's Medical Center, Minami-ku, Yokohama 232, Japan
Summary A female patient with unilateral gonadal dysgenesis was a mosaic for three cell lines, 45,X/46,X,+marI/46,X,+marII, including two different marker chromosomes. DNA analysis using 17 Y-specific DNA probes revealed that each marker consists of different segments of the Y chromosome. Key Words Y chromosome, marker chromosome INTRODUCTION
It is difficult to identify the origin of small marker chromosomes using cytological techniques as many marker chromosomes fail to show characteristic banding patterns. In the present report, we used 17 Y-specific DNA probes to analyze DNA from a female patient with clitoral enlargement who had two different marker chromosomes. Each of the markers consists of a part of the Y chromosome. CASE REPORT
A newborn girl was investigated, because of clitoral hypertrophy and bilateral inguinal hernias. The presence of vagina and uterus was established by the urethrography. Clitoroplasty and surgery to correct the bilateral inguinal hernias were Received March 2, 1991; revised version received May 1, 1991; Accepted May 7, 1991. 195
196
T. TAMURA et aL
performed. The right hernial content was proven to be testis and was excised. A uterus and a possible streak gonad were found in the left inguinal hernia and a small part of the latter was removed. At eight months of age, no development delay was apparent. Clinical features were normal except for those noted above. Laboratory analyses revealed: blood testosterone 0.9 ng/ml; elevated LH (282 ng/ml) and F S H (655 ng/ml), normal values being 0-1, 4-63 and 0-150 respectively; and normal adrenal function. CYTOGENETIC STUDIES A 4 5 , X / 4 6 , X , + m a r I / 4 6 , X , + m a r I l karyotype was observed in cells cultured from peripheral blood leukocytes (Fig. 1). Out of 31 cells karyotyped, five were 45,X, 23 had mar I and three had mar II. Both markers I and II were Q-negative. The former was a small ring chromosome (Fig. la). The latter was a G-sized chromosome. In some of mitotic spreads marker II appeared to be dicentric (Fig. lb). Additional material for a C-band study was not available. DNA STUDIES A total of 17 Y-specific D N A probes were used. Ten of them: 87-4(AMGL); 87-6(DYS135); 87-7(DYSI30); 87-17(DYS131); 87-19(DYS132); 87-24(DYS133); 87-26(DYS139); 87-27(DXYS73Y); 87-28(DYS140), and 87-31(DYS134), were cloned by Nakahori et al. (1991). The pHY10 probe detects the repetitive D N A family DYZ1 in the Q-positive region (interval 7) of the Y chromosome (Nakahori
Fig. 1. Two marker chromosomes in the patient. Top: Partial karyotype of a cell with marker I. From left; G stained chromosome pairs, numbers 21 and 22, an X chromosome and marker I. At the far right, the Q-stained marker is shown. Bottom: As above except that marker II instead of marker I is shown. Jpn. J. Human Genet.
TWO DIFFERENT MARKERS
50f2
197
pHY10
Fig. 2. Examples of Southern analysis. For each probe, both control male DNA and that of the patient are shown. In the 50f2 blot (left), arrow heads indicate loci A to E (from the top). Loci A, B and D showed decreased but distirlct signals ( + § in Fig. 3). The band corresponding to locus C of S0f2 barely distinguishable. The band corresponding to 50f2 locus E is not detectable under our standard hybridization conditions and was observed only after prolonged exposure (not shown). The patient is pHY10 negative (right). et al., 1986). The remaining six probes were gifts from Dr. J. Weissenbach (probes 47z, 52d, 50f2, 12f3, and 49f; Vergnaud et al., 1986) and a gift from Dr. D. C. Page (probe pDP1007(ZFY); Page et al., 1987). Probe 50f2 detects five different loci (50f2A to 50f2E) on the Y chromosome (see Fig. 2). Probe 52d detects three different loci: 52dA ( Y q l l ) ; 52dB (Yp) and 52dC (Yp). Probe 87-17 (DYS131) detects two different sites DYS131A (Yq) and DYS131B (Yp). The 17Y-specific D N A probes had already been mapped on the Y chromosome and were found to detect a total of 23 loci (Nakahori et aL, 1991). D N A was isolated from peripheral leukocytes by the standard technique (Maniatis et al., 1982). Probes pDP1007, 47z, 52d, 87-4, 87-7, 87-19, 87-26, and Vol. 36, No. 2, 1991
198
T. T A M U R A et al.
87-27 were used to probe Southern blots of TaqI digested patient DNA. The pHY10 probe was used in combination with StuI (Nakahori et aL, 1989). The remaining probes were used with EcoRI. Differences were observed between the patient DNA patterns of probes from separate regions of the Y chromosome. Firstly, the Y-specific repeated sequence DYZl identified by pHY10 was absent in the patient (Fig. 2). Second, the intensities of the signals produced, when the other probes were hybridized to the patient DNA, varied according to the location of those probes on the Y chromosome. Medium signal intensities were observed for probes between pDP1007 in interval 1 and 87-19 in interval 5-6. Low signal intensities were observed for probes between 87-6 and 49f in interval 5-6. The results of the DNA analysis are summarized in Fig. 3.
Probes/ loci pDP1007 47z 87-27 52dB 52dC 87-17B 50f2A,B
-IF "H-H44-H-IF -H-
50f2D
-IF
87-7 87-4 12f3 87-28 87-26 87-24 87-31 87-19 87-6 52dA 87-17A 50f2E 50f2C 49f pHYlO
+t-H4-I-i+ 44-H-H"H-t+ --t+ + +
Fig. 3.
1!
patient
Pseudoautosoma
1~ 2
i
"--] ,3~
n Sex9 . ~ IIFIK OQ
pDPlOO7
87-27(DXYS73Y) ;S~C~
5807;217B(DYS 131'
i~il " - ] 4 A J [--50,2D' ~ 1 I 1 8 7 - 7 (DYS130) ~ _ _ 1 4 B A--12f3 L_ 87_4 (AMGL) f~.:iiiil.---~ J ~87-28(DYS140) [~iiii] 1 5 , 87-26(DYS 139) [~iiit/I ~:
